CN117511862A - Adipose-derived mesenchymal stem cells for wound repair and culture method and application thereof - Google Patents
Adipose-derived mesenchymal stem cells for wound repair and culture method and application thereof Download PDFInfo
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- CN117511862A CN117511862A CN202311479764.9A CN202311479764A CN117511862A CN 117511862 A CN117511862 A CN 117511862A CN 202311479764 A CN202311479764 A CN 202311479764A CN 117511862 A CN117511862 A CN 117511862A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention belongs to the technical field of skin wound repair, and particularly relates to a adipose-derived mesenchymal stem cell for wound repair, a culture method and application thereof. The parameters of frequency, intensity, pulse time and stimulation time in the low-intensity pulse ultrasound (LIPUS) treatment are adopted to treat the adipose-derived mesenchymal stem cells (ADSCs), so that the biological response of the adipose-derived mesenchymal stem cells can be activated to the maximum extent, the survival rate, activity and functionality of the cells are improved, the biological processes of cell proliferation, angiogenesis, matrix synthesis and the like of a skin wound surface are accelerated, the healing of the wound surface is finally promoted, scar formation is reduced, and functional recovery is improved.
Description
Technical Field
The invention belongs to the technical field of skin wound repair, and particularly relates to a adipose-derived mesenchymal stem cell for wound repair, a culture method and application thereof.
Background
Wound repair is a critical topic in the medical field. Burns, wounds, diabetes, chronic ulcers, etc. often result in defects in the skin. Pain, loss of physiological function, and changes in appearance caused by intractable, chronic wounds and tissue defects seriously affect the freedom of movement and quality of life of patients, and further cause psychological and social problems. Traditional wound repair methods include suturing, skin flap grafting, skin grafting, and the like, but have limitations such as long-term healing, incomplete regeneration, scarring, and the like. Therefore, there is a need to develop new methods to promote the speed and quality of wound repair.
ADSCs (Adipose-derived mesenchymal stem cells, ADSCs) are a type of pluripotent stem cells with self-renewal and multipotent differentiation potential, which are present in Adipose tissue. ADSCs become a research hotspot in the fields of tissue engineering and regenerative medicine because of their abundant sources, ready availability and high proliferation capacity. LIPUS (Low-intensity pulsed ultrasound, LIPUS) is a non-invasive physical stimulation technique and has been widely used in the field of orthopedics and soft tissue repair. Researches show that the LIPUS can promote the processes of fracture healing, soft tissue repair, wound healing and the like. LIPUS accelerates tissue regeneration and repair by stimulating biological processes such as cell proliferation, differentiation, and matrix synthesis.
However, the existing method for stimulating the proliferation of ADSCs by using LIPUS has the problem of low survival rate of ADSCs after stimulation, and cannot better activate biological response of ADSCs, thereby affecting the functionality of ADSCs in the wound repair process.
Disclosure of Invention
The invention aims to provide a adipose-derived mesenchymal stem cell for wound repair, and a culture method and application thereof, wherein the culture method can better activate the biological performance of the adipose-derived mesenchymal stem cell, so that the obtained adipose-derived mesenchymal stem cell has higher survival rate and activity and better wound repair effect.
The invention provides a culture method of adipose-derived mesenchymal stem cells for wound repair, which comprises the following steps:
performing low-intensity pulse ultrasonic treatment on the adipose-derived mesenchymal stem cells to obtain the adipose-derived mesenchymal stem cells for wound repair;
the frequency of the low-intensity pulse ultrasonic treatment is 0.5-2 MHz, the duty ratio is 10-50%, the time is 1-20 min, and the intensity is 0.1-00W/cm 2 。
Preferably, the preparation method of the adipose-derived mesenchymal stem cells comprises the following steps:
soaking adipose tissues with normal saline containing antibiotics and serum, and then cleaning the soaked adipose tissues to obtain adipose tissue particles;
performing tissue digestion on the adipose tissue particles by using digestive enzymes to obtain a cell suspension;
and centrifuging the cell suspension, and washing the obtained cell sediment to obtain the adipose-derived mesenchymal stem cells.
Preferably, the antibiotic comprises penicillin-streptomycin double antibody, the mass concentration of the penicillin-streptomycin double antibody in the physiological saline containing the antibiotic and the serum is 1%, and the mass concentration of the serum is 1%.
Preferably, the digestive enzyme comprises collagenase type i; the enzyme activity of the collagenase I is more than or equal to 125CDU/mg; the final concentration of the collagenase I in a tissue digestion system is 0.5-2 mg/mL.
Preferably, the conditions of tissue digestion include: the temperature is 30-40 ℃, the oscillation frequency is 50-200 rpm, and the time is 0.5-1.5 h.
The invention also provides the adipose-derived mesenchymal stem cells for wound repair obtained by the culture method according to the technical proposal, and the cells are used for OD 450nm The cell survival rate of the adipose-derived mesenchymal stem cells for wound repair is more than or equal to 95 percent.
The invention also provides application of the adipose-derived mesenchymal stem cells for wound repair in preparing a product for skin wound repair.
Preferably, the product comprises a medicament.
The invention also provides a medicine for repairing the skin wound, and the active ingredients of the medicine comprise the adipose-derived mesenchymal stem cells for repairing the wound.
Preferably, the dosage form of the medicament comprises injection, spray or liniment.
The beneficial effects are that:
the invention provides a culture method of adipose-derived mesenchymal stem cells for wound repair, which comprises the following steps: performing low-intensity pulse ultrasonic treatment on the adipose-derived mesenchymal stem cells to obtain the adipose-derived mesenchymal stem cells for wound repair; the frequency of the low-intensity pulse ultrasonic treatment is 0.5-2 MHz, the duty ratio is 10-50%, the time is 1-20 min, and the intensity is 0.1-100W/cm 2 . The parameters of frequency, intensity, pulse time and stimulation time in the low-intensity pulse ultrasonic treatment are adopted to treat the adipose-derived mesenchymal stem cells, so that the biological response of the adipose-derived mesenchymal stem cells can be activated to the maximum extent, the survival rate, activity and proliferation functionality of the cells are improved, the biological processes of cell proliferation, angiogenesis, matrix synthesis and the like of a skin wound surface are accelerated, the healing of the wound surface is finally promoted, scar formation is reduced, and functional recovery is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a schematic diagram of the flow assay of adipose-derived stem cells in example 1;
FIG. 2 is a schematic diagram of the LIPUS stimulator device of example 1;
FIG. 3 is a graphical representation of results of a LIPUS-stimulated ADSCs condition screening assay in example 1;
FIG. 4 shows the results of the detection of the activity of hADSC-P3 by low intensity ultrasound treatment in example 1;
FIG. 5 is a schematic diagram of a wound repair procedure according to example 2;
FIG. 6 is a graph showing the comparison of wound closure times for the experimental and control groups of example 2;
FIG. 7 shows the results of the distribution of ADSCs in tissue according to example 2;
FIG. 8 is a schematic diagram showing histological analysis of wound surfaces of experimental group and control group in example 2 using Ki-67 as proliferation marker;
FIG. 9 is a schematic diagram showing histological analysis of wound surfaces of the experimental group and the control group in example 2, wherein CD31 is used as a proliferation marker;
FIG. 10 is a schematic diagram of Masson staining results of wounds of the experimental and control groups in example 2;
fig. 11 is a schematic diagram showing the results of wound inflammation analysis of the experimental group and the control group in example 2.
Detailed Description
The invention provides a culture method of adipose-derived mesenchymal stem cells for wound repair, which comprises the following steps:
performing low-intensity pulse ultrasonic treatment on the adipose-derived mesenchymal stem cells to obtain the adipose-derived mesenchymal stem cells for wound repair;
the frequency of the low-intensity pulse ultrasonic treatment is 0.5-2 MHz, the duty ratio is 20%, the time is 1-20 min, and the intensity is 0.1-100W/cm 2 。
The invention preferably prepares adipose-derived mesenchymal stem cells, and the preparation method of the adipose-derived mesenchymal stem cells preferably comprises the following steps:
soaking adipose tissues with normal saline containing antibiotics and serum, and then cleaning the soaked adipose tissues to obtain adipose tissue particles;
performing tissue digestion on the adipose tissue particles by using digestive enzymes to obtain a cell suspension;
and centrifuging the cell suspension, and washing the obtained cell sediment to obtain the adipose-derived mesenchymal stem cells.
The present invention preferably acquires adipose tissue. The adipose tissue of the invention preferably comprises sterile and high-activity (tissue activity is more than or equal to 95 percent) adipose tissue, and on the basis, the adipose tissue source preferably comprises adipose tissue of human or animal, and more preferably comprises adipose tissue obtained by liposuction operation; the adipose tissue obtained by the liposuction operation can achieve the effect of reutilizing waste adipose tissue and changing waste into valuable, and meanwhile, the adipose tissue from the source has wide material source, is convenient to obtain and has low immunogenicity and no immunological rejection. The specific procedure for obtaining adipose tissue is not particularly limited, and can be obtained according to the conventional procedures in the art, such as liposuction or direct excision.
After obtaining the adipose tissue, the present invention preferably immerses the adipose tissue with physiological saline containing antibiotics and serum to sterilize the adipose tissue. The antibiotics comprise penicillin-streptomycin double antibodies, wherein the mass concentration of the penicillin-streptomycin double antibodies in the physiological saline containing the antibiotics and the serum is preferably 1%, namely the mass concentration of the penicillin and the streptomycin in the physiological saline containing the antibiotics and the serum is preferably 1%; the concentration of serum in the physiological saline containing the antibiotic and serum is preferably 1%. The soaking time of the present invention is preferably 5 to 20 minutes, more preferably 15 minutes.
After the soaking, the invention preferably cleans the fat tissue obtained by soaking to obtain fat tissue particles. The washing is preferably performed with physiological saline; the washing step further comprises centrifuging the washed mixture to remove superfluous grease and waste liquid from the adipose tissues through repeated washing and centrifuging. The specific steps of washing and centrifuging are not particularly limited in the present invention, and may be carried out according to the conventional washing and centrifuging steps in the art.
After obtaining the adipose tissue particles, the present invention preferably performs tissue digestion on the adipose tissue particles with digestive enzymes to obtain a cell suspension. The digestive enzymes of the invention preferably comprise collagenase type i; the enzyme activity of the collagenase I is preferably more than or equal to 125CDU/mg, more preferably 100-150 CDU/mg; the final concentration of collagenase type I in the tissue digestion system is preferably 0.5-2 mg/mL, more preferably 1mg/mL. The temperature of tissue digestion according to the invention is preferably 30-40 ℃, more preferably 37 ℃; the oscillation frequency is preferably 50 to 200rpm, more preferably 100rpm; the time is preferably 0.5 to 1.5 hours, more preferably 1 hour.
After obtaining the cell suspension, the cell suspension is preferably centrifuged to obtain a cell pellet. The rotational speed of the centrifugation is preferably 1000-3000 g, more preferably 1500g; the time is preferably 5 to 20 minutes, more preferably 10 minutes.
After obtaining the cell pellet, the present invention preferably washes the cell pellet to obtain the adipose mesenchymal stem cells. The washing according to the present invention preferably includes mixing and centrifuging the cell pellet with physiological saline, repeating the washing 3 times, and removing impurities. The rotational speed of the centrifugation is preferably 1000-3000 g, more preferably 1500g; the time is preferably 5 to 20 minutes, more preferably 10 minutes.
The preparation method of the invention preferably further comprises the step of carrying out adherent culture on the adipose-derived mesenchymal stem cells so as to amplify the adipose-derived mesenchymal stem cells. The cell culture medium of the present invention for the adherent culture preferably comprises a low sugar medium, more preferably DMEM low sugar medium. The purity of the adipose-derived mesenchymal stem cells obtained by the adherent culture is preferably more than or equal to 95 percent. The specific conditions of the adherent culture are not particularly limited, and the conditions conventional in the art can be adopted.
After the adipose-derived mesenchymal stem cells are obtained, the invention carries out low-intensity pulse ultrasonic treatment on the adipose-derived mesenchymal stem cells to obtain the adipose-derived mesenchymal stem cells for wound repair. The frequency of the low-intensity pulse ultrasonic treatment is 0.5-2 MHz, preferably 1-1.5 MHz, more preferably 1.5MHz; the duty cycle is 10-50%, preferably 20%; the time is 1 to 20min, preferably 2.5 to 15min, more preferably 5 to 10min, most preferably 5min; the strength is 0.1-100W/cm 2 Preferably 25 to 50W/cm 2 More preferably 50W/cm 2 。
The low intensity pulsed ultrasound treatment according to the invention is preferably performed in the form of a adipose mesenchymal stem cell suspension, preferably a adipose mesenchymal stem cell PBS suspension. The cell concentration in the adipose-derived mesenchymal stem cell suspension of the present invention is preferably 1×10 7 /mL. In the low-intensity pulsed ultrasonic treatment according to the present invention, the probe of the low-intensity pulsed ultrasonic treatment apparatus is preferably connected with the device containing the adipose-derived mesenchymal stem cells by a coupling agent, and more preferably with the device containing the adipose-derived mesenchymal stem cellsThe bottom is connected to ensure the close contact between the low-intensity pulse ultrasonic treatment equipment and the adipose mesenchymal stem cell device. The invention has no special limitation on the model of the low-intensity pulse ultrasonic treatment equipment, and can ensure the equipment with the low-intensity pulse ultrasonic treatment parameters. The couplant of the present invention preferably comprises a medical gel ultrasound couplant.
The setting of the ultrasonic frequency, the intensity, the pulse duration and the stimulation time of the low-intensity pulse ultrasonic treatment realizes the accurate stimulation of the adipose-derived mesenchymal stem cells, the reasonable parameter design can activate the biological response of the adipose-derived mesenchymal stem cells, and the key processes of promoting the proliferation, the differentiation, the matrix synthesis and the like of the adipose-derived mesenchymal stem cells are improved.
The invention also provides the adipose-derived mesenchymal stem cells for wound repair obtained by the culture method according to the technical proposal, and the cells are used for OD 450nm The cell survival rate of the adipose-derived mesenchymal stem cells for wound repair is 90-99%, preferably 95%. The formula for calculating the cell viability according to the invention is preferably cell viability = [ (Experimental well OD) 450nm Blank well OD 450nm ) /(control well OD 450nm Blank well OD 450nm )]X 100%. The adipose-derived mesenchymal stem cells obtained by the method have multipotency and self-renewal capacity, can be differentiated into various cell types and participate in the process of tissue regeneration.
Based on the advantages, the invention also provides application of the adipose-derived mesenchymal stem cells for wound repair in preparing a product for skin wound repair. The product of the invention preferably comprises a medicament. The product of the invention can be applied to the skin wound surface in the form of injection, spray or smear; the administration dosage is preferably 10 6 ～10 7 Individual cells/wound surface with a diameter of 1 cm. The wound surface according to the present invention is preferably, but not limited to, a wound surface caused by burns, wounds, ulcers and surgical incisions. The application of the invention can directly apply the adipose-derived mesenchymal stem cells to the wound area, has non-invasive property, does not need to perform large-scale operation or incision, and reduces the sufferingThe pain and recovery time of the patient are high, and the safety is high; meanwhile, the directional application of the prepared adipose-derived mesenchymal stem cells for repairing the wound surface is realized, the local treatment effect on the wound surface is realized, and the pertinence and the effectiveness of treatment are improved; meanwhile, the application mode can be repeatedly used, and the medicine can be used and treated for multiple times according to the needs of patients, so that the sustainability and convenience of treatment are improved.
The invention also provides a medicine for repairing the skin wound, and the active ingredients of the medicine comprise the adipose-derived mesenchymal stem cells for repairing the wound. The concentration of the adipose-derived mesenchymal stem cells for wound repair in the medicament of the invention is preferably 1 multiplied by 10 6 ～1×10 7 Preferably 1X 10 per mL 7 /mL. The dosage forms of the medicament of the invention preferably comprise injection, spray or liniment. The invention has no special limitation on the types of auxiliary materials in the medicine, and the conventional auxiliary materials in the field can be selected according to the dosage form of the medicine.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
1: the acquisition and preparation of ADSCs comprises the following specific steps:
(1) obtaining ADSCs from adipose tissue of human origin: adipose tissue can be obtained by a liposuction operation method;
(2) soaking and sterilizing the fat component after impurity removal in physiological saline (containing penicillin-streptomycin double antibody with the mass concentration of 1 percent and the mass concentration of serum of 1 percent) for 15 minutes, and repeatedly washing and centrifuging the fat component with the physiological saline to remove redundant grease and waste liquid, wherein only fat tissue particles are reserved;
(3) placing the washed adipose tissue particles in the step (2) in a shaking table at 37 ℃ for shaking digestion for 1h at 100rpm, wherein the final concentration of collagenase I is 1mg/mL, and the enzyme activity of collagenase I is more than or equal to 125CDU/mg;
(4) centrifuging the suspension obtained in the step (3) under the condition of 1500g and 10min, and then removing the upper layer waste liquid to keep the bottom cell sediment;
(5) putting the cells obtained in the step (4) into a proper amount of physiological saline, fully re-suspending the cells, performing centrifugal operation for 1500g and 2min, and repeatedly cleaning for three times to remove impurities as much as possible;
(6) and (3) performing adherent culture and passage on the cells obtained in the step (5) by using a DMEM low-sugar culture medium to obtain high-quality and high-purity humanized ADSCs, wherein the purity of the obtained ADSCs is 99%.
The resulting ADSCs were subjected to flow assay analysis, the results of which are shown in FIG. 1. From fig. 1, it can be derived that: the results of the flow analysis of the prepared ADSCs show that the surface markers CD11b, CD34 and CD45 are expressed negatively, and CD44, CD90, CD29 and CD105 are expressed positively, and the results conform to the definition of the International cell Association on ADSCs, which indicates that the humanized ADSCs are successfully prepared.
2: stimulating ADSCs cells with LIPUS using an ultrasonic system (Sonic Accelerated Fracture Healing System, SAFHS), attaching a sensor probe to the bottom of a cell dish, coating a couplant on the bottom of the cell dish, adhering the two to make full contact with each other, and stimulating PBS suspension of ADSCs in the cell dish, wherein the LIPUS comprises an ultrasonic generator and an ultrasonic probe as shown in FIG. 2; wherein the volume of the PBS suspension of ADSCs is 200. Mu.L/dish, and the cell concentration is 1×10 7 /mL。
Physical intervention is carried out on cells by adjusting different ultrasonic parameters, and the set stimulation parameters are as follows: the frequency is 1.5MHz, the duty ratio is 20%, and the intervention time is respectively as follows: 0. 2.5, 5, 10min, ultrasonic intensities are respectively: 0. 25, 50, 100mW/cm 2 The optimal stimulation conditions were obtained by screening for each group of cell proliferative activities, the specific settings and results are shown in FIG. 3, wherein 24h and 48h in the first and second row graphs in FIG. 3 represent the measured cell activities of each group at 24h and 48h after stimulation, respectively.
As can be seen from FIG. 3, when the ultrasonic intensity is 25mW/cm 2 There was no change in cell activity upon increasing the stimulation time when the intensity of ultrasound was 100mW/cm 2 When the stimulation time is increased, the cell activity is gradually reduced, and only the ultrasonic intensity is obtained50mW/cm 2 When the stimulation is carried out for 5min, the cell activity reaches the optimum, and the cell activity is not increased any more after the stimulation is continued for a prolonged period of time, so that the optimum LIPUS stimulation condition is that the frequency is 1.5MHz, the duty ratio is 20%, the time is 5min, and the intensity is 50mW/cm 2 。
After treatment of ADSCs with the optimal parameters, cells were subjected to viable cell staining after 24h and 72h, respectively, as can be seen from FIG. 4, using 50mW/cm 2 The activity of the two groups of cells after 24h stimulation of the cells was significantly different, and the number of living cells in the 72h post-treatment group was significantly higher than that in the control group (0 mW/cm 2 ). This demonstrates that the optimal parameters obtained by screening help to increase cell activity.
Example 2
The wound repair experiment of the mouse experiment comprises the following steps:
the experiment was divided into four groups, 3 of which were control groups, 1 of which were experimental groups: PBS injection group, low dose ADSC group (10 6 ) And high dose ADSC group (10 7 ) Forming 3 control groups; low dose ADSC after LIPUS stimulation (10 6 ) As experimental groups. 6 columns of each group are repeated for 3 times, and wound skin repairing effects among the groups are observed; wherein the low dose group, the high dose group and the experimental group are injected in the form of cell PBS suspension, and the injection doses of the four groups are 200 mu L/wound surface with the diameter of 1 cm.
1: ADSCs are obtained and prepared in the same manner as in example 1.
2: the method comprises the following steps of:
healthy 6-week-old C57 mice were selected as subjects. The mice were shaved on their backs and the wound area was then cleaned and disinfected with sterile surgical instruments.
1) Anesthesia: the mice in the experimental group and the control group are respectively anesthetized by a proper amount of anesthetic, so that the mice are in a painless and non-resistant state;
2) Preparing a wound surface: manufacturing a wound surface with the diameter of about 1cm on the back skin of each mouse, wherein the depth reaches subcutaneous tissue, and the wound surface is circular so as to ensure the size and the position of the wound surface to be consistent;
3: the best LIPUS stimulation conditions (frequency) screened in example 1 were used1.5MHz, duty cycle 20%, time 5 minutes, intensity 50mW/cm 2 ) And (3) performing ultrasonic stimulation on the ADSCs in the step 1. And uniformly injecting each group of prepared ADSCs suspension into the wound surface area of the mice to ensure that the cell suspension fully permeates into the wound surface and ensure that cells are uniformly distributed.
1) Experimental group treatment: the mice of the experimental group were subcutaneously spot-injected with ADSCs, which were previously cultured by LIPUS treatment, using a sterile syringe, and gently massaged to ensure uniform distribution of cells on the wound.
2) Control group treatment: three control groups of mice were injected subcutaneously with the same volume of PBS solution as a control treatment.
3) Care and observation: all mice were properly attended, including wound cleaning and disinfection. And (5) observing and recording the healing condition of the wound surface every day, wherein the healing condition comprises indexes such as wound surface closing time, inflammatory response, redness and swelling degree, healing quality and the like.
4) Wound surface sample collection: wound tissue samples of mice in the experimental group and the control group were collected at predetermined time points (e.g., 1, 3, 7, 10 days) as shown in fig. 5 to 6, wherein adsc+li in fig. 5 represents the experimental group.
From fig. 5 to 6, it can be derived that: the wound closure time of the experimental group is obviously shortened, and the wound healing speed is obviously accelerated compared with the control group. The experimental group is obviously reduced at the beginning of the 7 th day, the difference has statistical significance, the wound surface is basically healed at the 10 th day, the other three groups only have no significant difference compared with the experimental group at the 7 th day, and the wound surface sizes of the other three groups have significant differences compared with the experimental group at the 10 th day. Therefore, the LIPUS provided by the invention can obviously promote the repair of the wound surface of the skin after injury by stimulating ADSCs.
4: the ADSCs are marked by using fluorescent dye PKH26 and then wound surface treatment is carried out, the skin tissues near the wound surface are obtained after 3 days, the fixation and distribution of the ADSCs in the animal skin tissues are observed by using a fluorescence microscope, and as shown in figure 7, ADSCs (red) cells are uniformly distributed in tissues below the dermis layer of the skin tissues, thereby demonstrating that the ADSCs can stably exist in subcutaneous tissues and can play a role in treatment subsequently.
5: the wound samples were subjected to histological analysis, immunohistochemical staining and molecular biological detection with reference to Liang, X., lin, F., ding, Y.et al.conditioned medium frominduced pluripotent stem Cell-derived mesenchymal stem cells accelerates cutaneous wound healing through enhanced, anatomy, stem Cell Res Ther 12,295 (2021), https:// doi.org/10.1186/s13287-021-02366-x, to assess cellular and molecular changes in wound repair. Wherein the molecular biological assay is a fluorescent quantitative PCR assay (Q-PCR), and wherein the primers used for the Q-PCR are as shown in Table 1, with GAPDH as an internal reference gene.
TABLE 1Q PCR analysis primers
| Primer(s) | Sequence (5 '-3') | Sequence numbering |
| m-GAPDH-F | TCAATGAAGGGGTCGTTGAT | SEQ ID NO.1 |
| m-GAPDH-R | CGTCCCGTAGACAAAATGGT | SEQ ID NO.2 |
| m-IL-6-F | TGGTACTCCAGAAGACCAGAGG | SEQ ID NO.3 |
| m-IL-6-R | AACGATGATGCACTTGCAGA | SEQ ID NO.4 |
| m-TNF-α-F | GGTCTGGGCCATAGAACTGA | SEQ ID NO.5 |
| m-TNF-α-R | CAGCCTCTTCTCATTCCTGC | SEQ ID NO.6 |
The results of the histological analysis are shown in fig. 8 to 9. From fig. 9, it can be derived that: a significant increase in the number of positive cells for proliferation markers such as Ki-67 was detected by immunohistochemical staining. Furthermore, it can be seen from fig. 8 that angiogenesis was also enhanced in the experimental group, and more angiogenesis was observed by CD31 staining. The application of ADSCs is shown to promote wound tissue regeneration and angiogenesis.
The immunohistochemical staining results are shown in FIG. 10, wherein ADSC-LIPUS in FIG. 10 represents the experimental group. The Masson staining results in fig. 10 show that the expression level of matrix components such as collagen and elastin is significantly increased in the experimental group, which indicates that the LIPUS stimulation promotes the matrix reconstruction process of the wound tissue, and the wound tissue in the experimental group shows better matrix reconstruction capability.
From fig. 11, it can be derived that: compared with the control group, the wound tissue inflammatory reaction of the experimental group is obviously reduced. Q-PCR results showed that inflammatory cell infiltration and inflammatory factors such as IL-6 and TNF- α expression were significantly reduced in the experimental group, indicating that LIPUS stimulation helped inhibit the inflammatory response of the wound.
From the above results, it can be derived that: ADSCs cells for repairing the skin wound are prepared by stimulating the ADSCs through the LIPUS, and experiments prove that the ADSCs cells for repairing the skin wound can remarkably accelerate the healing of the wound and the tissue regeneration; meanwhile, the method disclosed by the invention is non-invasive and low in risk, avoids the problems of wounds, infection and rejection reactions possibly existing in the traditional method, and is safer and more effective.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. A method for culturing adipose-derived mesenchymal stem cells for wound repair, comprising:
performing low-intensity pulse ultrasonic treatment on the adipose-derived mesenchymal stem cells to obtain the adipose-derived mesenchymal stem cells for wound repair;
the frequency of the low-intensity pulse ultrasonic treatment is 0.5-2 MHz, the duty ratio is 10-50%, the time is 1-20 min, and the intensity is 0.1-100W/cm 2 。
2. The culture method according to claim 1, wherein the method for preparing adipose-derived mesenchymal stem cells comprises the steps of:
soaking adipose tissues with normal saline containing antibiotics and serum, and then cleaning the soaked adipose tissues to obtain adipose tissue particles;
performing tissue digestion on the adipose tissue particles by using digestive enzymes to obtain a cell suspension;
and centrifuging the cell suspension, and washing the obtained cell sediment to obtain the adipose-derived mesenchymal stem cells.
3. The method according to claim 2, wherein the antibiotic comprises penicillin-streptomycin diabody, and the mass concentration of penicillin-streptomycin diabody in physiological saline containing the antibiotic and serum is 1%, and the mass concentration of serum is 1%.
4. The method of claim 2, wherein the digestive enzyme comprises collagenase type i; the enzyme activity of the collagenase I is more than or equal to 125CDU/mg; the final concentration of the collagenase I in a tissue digestion system is 0.5-2 mg/mL.
5. The culture method of claim 2 or 4, wherein the conditions of tissue digestion comprise: the temperature is 30-40 ℃, the oscillation frequency is 50-200 rpm, and the time is 0.5-1.5 h.
6. A adipose-derived mesenchymal stem cell for wound repair cultured by the culture method according to any one of claims 1 to 5, wherein the cells have an OD 450nm The cell survival rate of the adipose-derived mesenchymal stem cells for wound repair is more than or equal to 95 percent.
7. The use of adipose-derived mesenchymal stem cells for wound repair according to claim 6 for preparing a product for skin wound repair.
8. The use of claim 7, wherein the product comprises a medicament.
9. A medicament for skin wound repair, characterized in that the active ingredient of the medicament comprises the adipose-derived mesenchymal stem cells for wound repair according to claim 6.
10. The medicament according to claim 9, wherein the dosage form of the medicament comprises an injection, a spray or a liniment.
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